RAR agonist enhances EGCG-induced cell growth inhibition through 67LR upregulation
To investigate the participation of RAR in ATRA-mediated enhancement of EGCG-induced cell growth inhibition through 67LR, B16 cells were treated with the pan-RAR agonist TTNPB. TTNPB enhanced the protein levels and cell-surface levels of 67LR after 48 h of treatment (Fig. 5A, B). Moreover, treatment with TTNPB enhanced EGCG-induced cell growth inhibition (Fig. 5C). The growth-suppressive effect by combination treatment with EGCG and TTNPB was obviously reduced upon treatment with anti-67LR antibody (Fig. 5D). These results suggest that RAR-mediated action is involved in the ATRA-induced enhancement of EGCG-elicited cell growth inhibition.
10.1371/journal.pone.0011051.g005 Figure 5  RAR agonist induced EGCG-elicited cell growth inhibition through 67LR upregulation.
A) Cells were treated with or without 0.1 µM ATRA or 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h and were analyzed by Western blot analysis. Levels of 67LR expression were normalized to β-Actin. Band intensities were quantified using NIH Image J software. B) Anti-67 LR antibody conjugated with Alexa Fluor 488 was used at a dilution of 1∶100. Photographs were taken under Keyence BZ-8100 fluorescence microscope. C) Cells were treated with 0.1 µM ATRA or RARα agonist, 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h, then treated with 0.5 µM of EGCG for 48 h. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at **p<0.01, ***p<0.001. D) B16 cells were treated with 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h, then the cells were treated with either anti-67LR (MLuC5) or control antibody (mouse IgM) for 2 h, and the cells were added 0.5 µM of EGCG for 48 h. Cells proliferation was assessed by the WST-1 reagent. Cell number was measured as 430 nm absorbance and shown as relative of control. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at ***p<0.001.